Electrophoresis gel migration apparatus

ABSTRACT

A gel capillary electrophoresis apparatus has gel capillaries (2) filled with gel (2a) that are fixed at both ends thereof on an upper plate 5 and a lower plate (6). The gel capillaries (2) are arranged coarse on the upper plate (5) for sample injection and dense on the lower plate (6) for fluorescence detection. The apparatus is made easy in the sample injection and high in the fluorescence detection efficiency so that throughput of analysis of DNA and the like can be increased, and is available for three-dimensional electrophoresis.

This application is a continuation-in-part of application Ser. No.07/942,605, filed Sep. 10, 1992 now U.S. Pat. No. 5,277,780.

BACKGROUND OF THE INVENTION

The present invention relates to an electrophoresis gel migrationapparatus for electrophoresis separation of a DNA or protein.

Conventionally, base sequence of a DNA has been determined in the waythat the DNA was labeled by a radio isotope element and subjected to theelectrophoresis gel separation before the separation pattern wastransferred onto a film. However, the prior art technique mentionedabove has the disadvantage that it is not only troublesome to use theradioactive label, but also it needs too much labor and time. Toovercome such problems, a new real-time fluorescent label method hasbeen recently used, as disclosed in the Japanese Patent ApplicationLaid-Open 61-62843.

The fluorescent label method mentioned above uses slab gel, while afurther new gel capillary electrophoresis method is now attractingattention. The gel capillary electrophoresis method provides ahigh-speed, high-sensitive analysis with use of a capillary filled withgel (hereinafter referred to as the gel capillary), as disclosed in theAnalytical Chemistry, vol. 62, pp. 900-903, 1990.

The gel capillary electrophoresis is ordinarily made in the way that onecapillary tube and a detection lens are built in a package to integrate.The capillary tube can be used repeatedly. However, it is usuallydiscarded whenever it is used a few times as the gel is distorted duringthe operation. To make possible analysis of many samples at a time,there has been a disclosure that a multiple of gel capillaries arearranged for measurement. In the measurement, the multiple of gelcapillaries are retained by the respective holders as disclosed in theBioTechniques, vol. 9, p. 74, 1990.

Any of those measurements having the capillary gel is made in the waythat light is irradiated around the end of the capillaries to excite thefluorescent-labeled DNA passing there to emit fluorescence for detectionof sample fragments. To make measurement at a high sensitivity andaccuracy, the capillaries have to be set precisely.

In order to measure many DNA samples at a time to increase throughput,numbers of gel capillaries have to be arranged. The gel capillaries haveto be replaced after a few times of measurement. This means that it mustbe easy to attach or detach the numbers of gel capillaries and to aligntheir positions. The injection of sample into the gel capillaries hasbeen made with the use of electric field into the ends of the gelcapillaries put in sample wells. However, no reports have been made forgood workmanship of injecting the sample if the numbers of gelcapillaries should be arranged. This is one of the problems to besolved.

Distances of the gel capillaries should be longer with respect toeasiness of the sample injection. But, they should be shorter forefficient measurement or the sample fragments. It therefore has beenneeded to develop an apparatus meeting the both requirements.

SUMMARY OF THE INVENTION

In view of the foregoing, it is an object of the present invention toprovide a gel capillary electrophoresis separation portion integrated insuch a way that numbers of gel capillaries are coupled at both endsthereof with sample well side and a detection end for detection ofsample fragments, in order to make easy attachment or detachment andalignment of the gel capillaries. It is the other object of the presentinvention to provide a highly sensitive gel capillary electrophoresisapparatus providing good workmanship of sample injection.

Briefly, the foregoing objects are accomplished in accordance withaspects of the present invention by a gel capillary electrophoresisapparatus. The apparatus is of gel capillary cartridge type that, asshown in FIGS. 1a and 1b, can separate the gel capillary electrophoresisseparation portion from a sample injection plate 3 having sample wells 4to be injected with samples and a detector portion 7 for detectingfluorescent light from sample fragments. The apparatus includes a gelcapillary cartridge 1 having, in combination, an upper plate 5 of widearea, the gel capillaries 2, and a lower plate 6 of narrow area. Theupper plate 5 couples the gel capillaries 2 with the sample injectionplate 3 at capillary terminuses thereof from which the samples areinjected. The lower plate 6 couples the gel capillaries 2 with thedetector portion 7 at detection ends at which the samples migrated aredetected. The gel capillary cartridge 1 can be replaced with everymeasurement. This does not only increase throughput, but also makes thesample injection easy for simple setting of the gel capillaries 2. Thegel capillaries 2 are made dense on the narrow area of the detectorportion 7 so that any of the lights emitted from the sample fragmentscan be focused on an high-sensitivity image sensor on the detectorportion 7 without shrinking any of remaining image. The detector portion7 thus provides a highly efficient photodetection.

The gel capillary cartridge 1 can practically hold 20 to 500 gelcapillaries 2 so that the throughput of the sample measurement can beincreased. The sample injection can be made easy in the way that the gelcapillaries 2 are made coarse at the ends arranged and fixed on theupper plate 5 of the gel capillary cartridge 1 and the ends are alignedwith a sample holder or injection jig. The gel capillary electrophoresiscan be made high in sensitivity as the detector portion 7 can beincreased in the detection efficiency in the way that the gelcapillaries 2 are made dense at the other ends fixed on the lower plate6.

The capillaries available for the gel capillaries 2 are not limited intheir inside diameter, wall thickness, and length. The inside diametershould be smaller than 0.3 mm, ordinarily 0.1 to 0.2 mm, for convenienceof bending. The wall thickness should be usually made 0.1 to 0.2 mm. Thelength should be practically 10 to 100 cm, ordinarily 30 to 50 cm.Outside diameter of the capillaries should be ordinarily made 0.3 to 0.4mm.

The detector portion 7 has a gap between bulkheads 7a and 7b provided inparallel, the gap width being virtually equal to the inside diameter ofthe capillaries. The gap is filled with buffer solution or gel. Thebuffer solution or gel is irradiated by an excitation light. Theexcitation light has to be made to irradiate all the migration paths atthe same time. If the excitation light is not scanned, it cannotdirectly irradiate all the capillary tubes at a time. For the reason,the excitation light should irradiate positions at which the sampleselute from the capillaries. If the irradiation positions are too closeto the lower ends of the capillaries, scattering at the ends of thecapillaries affect the measurement. If they are too far from the lowerends, on the other hand, the samples eluted may mix with one another.Both cases are undesirable. In the present invention, it is effectivethat the excitation light should irradiate positions 0.5 to 1 mm awayfrom the lower ends of the capillaries. If the excitation light, such aslaser beam, is scanned for measurement, it may irradiate the capillarytubes themselves because diverged light beams having passed through thecapillary tubes are not re-used. It is good that distance between thebulkheads should be virtually equal to the inside diameter of thecapillaries of 0.1 to 0.2 mm.

The upper ends of the gel capillaries 2 can be coarse that is spaced notto disturb injection of the samples, say, intervals of 2 to 10 mm. Thelower ends should be as dense as possible to increase the fluorescencedetection efficiency, say 0.3 to 1 mm. The densities however are limitedto those.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention may be more fully described by reference to theaccompanying drawings in which:

FIG. 1a is a schematic view of major parts of an electrophoresis gelmigration apparatus of an embodiment of the present invention;

FIG. 1b is a cross-section of a portion (a plane passing a center of anyof gel capillaries on a sample injection plate) around a sampleinjection portion of the electrophoresis gel migration apparatus of theembodiment;

FIGS. 2a, 2b, and 2c are cross-sections on a plane and its variationspassing the center of any of the gel capillaries around a fluorescencedetector portion of the embodiment;

FIG. 3a is a cross-section of the electrophoresis gel migrationapparatus of the embodiment of the present invention;

FIG. 3b is a cross-section of a variation of a portion around thefluorescence detector portion of the embodiment;

FIG. 4 is a schematic view for major parts of an electrophoresis gelmigration apparatus of another embodiment of the present invention inwhich lower ends of the gel capillaries are arranged in two dimensionson the detector portion; and,

FIG. 5 is a cross-section of a portion around a fluorescence detectorportion of another embodiment.

DESCRIPTION OF THE PREFERRED EMBODIMENTS Embodiment 1

The following describes an embodiment 1 of the electrophoresis gelmigration apparatus according to the present invention by referring toFIGS. 1a, 1b, 2a, 2b, 2c, 3a, and 3b. FIG. 1a is a perspective viewillustrating major parts of a gel capillary electrophoresis migrationapparatus in the embodiment 1.

There is provided a gel capillary cartridge 1 in the apparatus. An upperplate 5 of the gel capillary cartridge 1 is coupled with a sampleinjection plate 3. Sample wells 4 on the sample injection plate 3 areimmersed in an upper buffer solution 18 in an upper buffer vessel 17shown in FIG. 3a. A lower plate 6 of the gel capillary cartridge 1 iscoupled with a detector portion 7 that can detect light emitted bysample fragments being eluted from a gel 2a in the gel capillaries 2 ofthe gel capillary cartridge 1. Distance between the upper plate 5 andthe lower plate 6 can be changed. In order to protect the gelcapillaries 2 from crash and to mechanically reinforce the gel capillarycartridge 1, however, the upper plate 5 and lower plate 6 are tiedtogether at their sides with a plastic ribbon (not shown) of apredetermined length. The plastic ribbon is a sheet-like ribbon ofaround 0.5 mm thick, around 2 cm wide, and 20 to 30 cm length. The gelcapillaries 2 are made of silica covered with polyimid resin on itssurface. The gel capillaries 2 can be bent as they are thin in thediameter and has the polyimid covered on the surface. The gelcapillaries 2 are bonded to the upper plate 5 and lower plate 6 as shownin FIG. 3a or are fixed with rubber rings 14 for holding capillary tubesas shown in FIG. 2a, with the upper plate 5 and sample injection plate 3mechanically aligned with faucet joint and screwed together. Similarly,the lower plate 6 and a detector portion 7 are connected together.

The electrophoresis gel migration apparatus can be made small as numbersof the gel capillaries 2 can be bundled together and bent so that theycan be contained in a narrow space for their long gel capillarymigration paths. For the accommodation, it is effective that the numbersof the gel capillaries 2 should be gathered in a way that the gelcapillaries 2 are sandwiched between two sheets of polymer film. In theway, if the gel capillary migration path used is 50 cm long, forexample, an electrophoresis plate needed is around 60 cm long for slabgel. The gel capillaries 2 then can be bent to a length shorter than 20cm for accommodation.

The upper plate 5, as described above, is tightly coupled with thesample injection plate 3 having sample wells 4 of 0.3 mm diameter, fourin a column thereof and 25 in a row thereof at a pitch of 5 mm. It isdesigned that each of the gel capillaries 2 and their respective samplewells 4 should be aligned. The upper plate 5, as described above, hasupper ends of the gel capillaries 2 arranged roughly at thereon in thetwo dimensions of columns and rows at the 5 mm pitch, while the lowerplate 6 has the lower ends aligned closer than the above on a straightline at a pitch of 1 mm. The lower plate 6 is attached to and coupledwith detector portion 7. The gel capillary cartridge 1 can be attachedwith or detached from the sample injection plate 3 and detector portion7. Couplings of the gel capillaries 2 with the lower plate 6 areprotected by rubber rings 14 for holding capillary tubes as shown inFIG. 2a. Similarly, couplings of the gel capillaries 2 with the sampleinjection plate 3 are protected by the rubber rings 14 for holdingcapillary tubes. The rubber rings 14 for holding capillary tubes areignored and not shown in FIGS. 1b, 2b, 2c, 3a, 3b, and 5. The rubberrings 14 for holding capillary tubes can be made of teflon or rubber aswell. A DNA sample that has been separated by the gel capillaries 2 thatis an electrophoresis separator of the sample, is eluted from gel 2a inthe gel capillaries 2 and enters the detector portion 7. FIG. 1b is across section A of FIG. 1a in the vicinity of the sample injection plate3. As any of the gel capillaries 2 is coupled with the upper plate 5,the sample injected into the corresponding one of the sample wells 4contacts the gel 2a. The gel capillaries 2 thus can be immersed in theupper buffer solution 18 as shown in FIG. 3a.

The embodiment 1 uses a sample adjusting titer plate having holes of 3mm diameter aligned at the same intervals of as the sample injectionplate 3 in addition to the sample injection plate 3 to inject the gel 2ainto the sample. The titer plate has thin silicon rubber film lined on abottom thereof. The titer plate is laid on the sample injection plate 3.The silicon rubber film can be broken with a needle or the like to makeholes of around 0.5 mm diameter. In this way, the sample in the holes ofthe titer plate can be easily injected into the gel 2a.

The upper plate 5 of the gel capillary cartridge 1 in the embodimentdescribed so far has the upper ends of the gel capillaries 2 arranged inthe two dimensions, but may have them in on dimensions, or in a straightline.

The detector portion 7 is coupled with the lower plate 6 of the gelcapillary cartridge 1 as shown in FIGS. 2a, 2b, or 2c which is a crosssection B of FIG. 1a. The migrated DNA sample elutes from the gel 2abefore migrating in a lower buffer solution 7d or a hollow portionfilled with the gel. An excitation light 8a is irradiated to excite afluorescence label of the DNA sample in a direction parallel with theline of the lower ends of the gel capillaries 2. The hollow portion thatis an irradiation light path for the excitation light 8a is formed ofbulkheads 7a and 7b of two silica plates. In the detector portion 7, asshown in FIGS. 2a and 2b, the excitation light source 8 for exciting thefluorescence label irradiates at a position around 0.5 mm in front ofthe lower ends of the gel capillaries 2 in a gap of 0.1 mm formed by thebulkheads 7a and 7b of the two silica plates placed in parallel. FIG. 2cis a variation of the example in FIG. 2b that the lower buffer solution7d or the gel can be easily immersed and contact the lower ends of thegel capillaries 2 from which the migrated DNA sample. The gap mentionedabove is filled with the lower buffer solution 7d or the gel and servesas the path for the excitation light 8a. The excitation light 8a of theexcitation light source 8, for example, a laser beam, reflected by areflection mirror 9 can irradiate at the position around 0.5 mm abovethe lower ends of the gel capillaries 2 so that it can irradiate the DNAsample eluting from all the gel capillaries 2 at substantially the sametime. So that in FIGS. 2b and 2c, the excitation light 8a is irradiatedin a direction perpendicular to the drawing.

A number of the gel capillaries 2 used in the embodiment 1 is 100.Fluorescent signals can be obtained from an range of around 10 cm on thebasis of the elution of the DNA sample as the excitation light 8a isirradiated. The fluorescent signals are detected by a photodetector 11,such as a line sensor, through a lens system 10 and a filter (not shown)at substantially the same time. The detected fluorescent signals areprocessed by a data processor 12 before fed out to an output device 13,such as a display.

If directly irradiated to the gel capillaries 2, the excitation light 8a(the laser beam here) is diverged, so that it cannot irradiate thenumber of the gel capillaries 2 at the same time. To solve such adifficulty, there can be a method that portions of the gel capillaries 2to be irradiated are immersed in the lower buffer solution 7d to makediffraction differences little so that scatter of the light at the tubeinterface of the gel capillaries 2 as the excitation light 8a isirradiated for the detection of the fluorescent signals. It, however, isnot always sufficient. There could be a better method that the detectorportion 7 has no capillary tubes provided therefor. In the embodiment 1,the DNA sample is eluted from the gel capillaries 2, and the excitationlight 8a is irradiated in a state of no capillary tubes or a statesimilar to it before the fluorescent signals are detected.

As shown in FIG. 3a, a detector is kept in a lower buffer vessel 7c, anda voltage is applied between an upper electrode 19 and a lower electrode20 in the upper buffer vessel 17 filled with the upper buffer solution18 before migration starts. The lower part of the electrophoresis gelmigration apparatus shown in FIG. 3a can be modified as shown in FIG.3b. In FIGS. 3a and 3b, the excitation light 8a for exciting thefluorescence label is irradiated in a direction perpendicular to thedrawing so that the DNA sample eluting in the path for the excitationlight 8a can generate fluorescent light. The fluorescent light isdetected in a direction C or C' of a plane formed of transparent silicaor a direction D. If the fluorescent light is detected in the directionC or C', for example, a fluorescent image of around 10 cm long is madesmall to one by four before detected by an image line sensor, forexample, of 25 mm long of the S3902, the Hamamatsu Photonix Inc., adiode array equipped with an image amplifier, or a CCD detector. If thefluorescent image is detected in the direction C shown in FIG. 3b, ithas the advantage that it has less effect due to reflection of the lightby the surface than the one C in FIG. 3a.

Embodiment 2

In turn, the following describes an embodiment 2 of the presentinvention by referring to FIG. 4. In the embodiment 2, lower ends of agel capillaries 2 are arranged and fixed in two dimensions of columnsand rows on a lower plate 6 of a gel capillary cartridge 1 so thatnumbers of the lower ends of the gel capillaries 2 can be collected in anarrow area. DNA sample eluting from the lower ends of the gelcapillaries 2 are detected by a two-dimensional detector 7. In thefigure, the lower ends of the gel capillaries 2 are arranged and fixedat a pitch of 1 mm in the columns and rows on the lower plate 6. Thedetector portion 7 has a group of detectors any of which is constructedas shown in FIG. 2a or 2b. That is, as shown in FIG. 5, an excitationlight 8a irradiates at a position around 0.5 mm below the ends of thegel capillaries 2 in a space filled with a lower buffer solution 7d orgel in a direction perpendicular to the drawing. In the embodiment 2,bulkheads 30a through 30z for forming the space of around 0.1 mm gap aspaths for the excitation light 8a may be non-transparent as lightsgenerated from the DNA sample can be detected below the detector portion7. The detector portion 7 is provided in the lower buffer solution 7d orthe gel in a lower buffer solution 7d. The excitation light 8a isreflected by a reflection mirror 9 to irradiate below the all the lowerends of the gel capillaries 2 in the lower buffer vessel 7c (not shownin FIG. 4) at substantially the same time by means of prisms 16a and 16bprovided one on each sides of the detector portion 7. Fluorescentsignals from DNA samples eluted from the gel capillaries 2 are alldetected at substantially the same time by the two-dimensional detectorportion 7 through an image reflection mirror 15, a lens system 10, and afilter (not shown). A bottom plate of the lower buffer vessel 7c (notshown in FIG. 4) is made of silica plate. Of course, for example, thereflection mirror 9 can be moved to sequentially scan the lineexcitation light 8a over the space around the area from one side of thedetector portion 7 to a point at which the lower buffer solution 7d orthe gel is filled with and the DNA sample elutes. Alternatively, thereflection mirror 9 can be made to irradiate at the same time the wholeline of the space around the area from the one side of the detectorportion 7 to the point at which the lower buffer solution 7d or the gelis filled with and the DNA sample elutes. The prisms 16a and 16b can beprovided either on an inside or the outside of the lower buffer vessel7c (not shown in FIGS. 4 and 5).

Note that parts of the electrophoresis gel migration apparatus above thegel capillaries 2 are not shown in FIG. 4.

In the embodiments described so far, the upper plate 5 of the gelcapillary cartridge 1 and the sample injection plate 3 can be attachedor detached together. The lower plate 6 of the gel capillary cartridge 1and the detector portion 7 also can be attached or detached together.Alternatively, the upper plate 5 and the sample injection plate 3 can beintegrated and the lower plate 6 and the detector portion 7 can beintegrated, and the two integrated couples can be assembled together tomake another form of gel capillary cartridge. More alternatively, onlyeither of the two couples mentioned above can be integrated to makestill another form of gel capillary cartridge. If the sample injectionplate 3 and the detector portion 7 are integrated with the gel capillarycartridge 1, the gel capillary cartridge 1 can be attached with ordetached from the upper buffer vessel 17 and the lower buffer vessel 7cat ends of the sample injection plate 3 and the detector portion 7,respectively.

In usual electrophoresis (one-dimensional electrophoresis), the sampleis separated in one-dimensional way in a direction x. In the so-calledtwo-dimensional electrophoresis, on the other hand, the separated samplehas an enzyme poured thereon to make some action, is separated again ina direction (direction y) perpendicular to the direction x to develop inthe two dimensions. The two-dimensional electrophoresis provides moredetailed separation of the sample than the one-dimensional one. It mayoccur that even the two-dimensionally separated pattern on the slab gelis lack of amount of information. In this case, it is effective that theslab gel having the sample separated thereon should be divided intonumbers of sections, the sample contained in each of the sections shouldbe made to act with enzyme or a DNA probe or the like, and a productmade through the action should be gel-separated again to obtain moreinformation. For the third electrophoresis separation, the presentinvention can use a capillary array distributed in two dimensions. Itcan be regarded as a separation in a direction z in relation to the onesin the directions x and y. The final information, or the third dimensioninformation, can be obtained in terms of a time-varying signal frommeasuring point distributed in the two dimensions. Alternatively, it canbe obtained in a way that the capillaries should be rearranged in aline, the signals should be obtained, and then data should be stored asif they were obtained in the two-dimensional arrangement.

It is not practical to employ a usual three-dimensional electrophoresishaving flat or block gel used therein, as the gel cross sections are toowide, allowing overcurrent to flow. In the gel capillaryelectrophoresis, current flowing through each of the gel capillaries isso small that no problems can be due to heat generation.

For the purpose of illustration only, the parts of the apparatus shownin FIGS. 1 to 5 are drawn different from those of the actual apparatusin proportions of shapes. The features of the present invention are asfollows. The electrophoresis gel migration apparatus according to thepresent invention can increase throughput to a great extent as it canhave numbers of gel capillary migration paths incorporated in the narrowdetection end area without affecting sample injection. Also, it canreduce sample injection work to a great extent as the arrangement of thesample wells on the sample injection plate are matched with that of thesample holes on the titer plate. Further, the apparatus can be madesmall as the gel capillaries can be bent so that the long gel capillarymigration paths can be incorporated in the narrow space. If the gelcapillary migration paths used are 50 cm long, for example, the slab gelrequires a migration plate of around 60 cm long, resulting in a largescale apparatus, while the gel capillaries can be bent to within 20 cmto contain. In order to have the migration paths as much as 100, theslab gel requires the migration plate of 40 cm wide or more, while thetwo-dimensional gel capillary arrangement allows the detector area to bemade as narrow as 1×1 cm or less.

What is claimed is:
 1. An electrophoresis gel migration apparatuscomprising gel capillaries being filled with gel, an excitation lightsource, fluorescent light detecting means, and means applying anelectric field to the gel; characterized in that the gel capillaries arearranged to provide a density of sample introduction ends of the gelcapillaries that is less than that of fluorescence detecting ends of thegel capillaries.
 2. An electrophoresis gel migration apparatus accordingto claim 1, characterized in that sample introduction ends of the gelcapillaries are arranged in two dimensions.
 3. An electrophoresis gelmigration apparatus according to claim 1, characterized in that thefluorescence detecting ends of the gel capillaries are arranged in onedimension.
 4. An electrophoresis gel migration apparatus according toclaim 1, characterized in that the sample having migrated through thegel capillaries elutes into a gap filled with buffer solution or the gelbefore being detected.
 5. An electrophoresis gel migration apparatusaccording to claim 4, characterized in that the sample introduction endsare arranged side-by-side in laterally spaced rows and the fluorescentdetecting ends of the gel capillaries are arranged linearly in a row oneafter the other, said excitation light source being arranged toirradiate all of the migrating samples at positions at which the sampleselude from the capillaries into said gap filled with buffer solution orthe gel substantially simultaneously.